LOCUS       sequence-347566-        6526 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses recombinant HyPro enzyme in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6526)
  AUTHORS   Yap K, Chung TH, Makeyev EV
  TITLE     Hybridization-proximity labeling reveals spatially ordered 
            interactions of nuclear RNA compartments.
  JOURNAL   Mol Cell. 2021 Nov 2. pii: S1097-2765(21)00838-8. doi: 
            10.1016/j.molcel.2021.10.009.
  PUBMED    34741808
REFERENCE   2  (bases 1 to 6526)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2021 Nov 2. pii: S1097-2765(21)00838-8. doi: 
            10.1016/j.molcel.2021.10.009."
COMMENT     Sequence Label: pML433
FEATURES             Location/Qualifiers
     source          1..6526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     939..958
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(978..1055)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1261..1280
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1364..1383
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1364..1382
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1383..1407
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1422..1444
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1452..1484
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             2727..2747
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2754..2771
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2824..2842)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2838..2885
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2922..3377
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3009..3028)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3219..3240
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3469..4284)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3469..4284)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4192..4211
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4276..4295
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4406..4994
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4895..4914
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5148..5165
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5180..5319
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5245..5264)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5405..5427
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5421..5612)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6421..6526,1..977))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
       61 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
      121 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
      181 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
      241 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
      301 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
      361 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
      421 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
      481 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      541 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
      601 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      661 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      721 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      781 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      841 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      901 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      961 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     1021 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     1081 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     1141 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     1201 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     1261 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     1321 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     1381 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1441 gagatataca tatggctagc atgactggtg gacagcaaat gggtcgcggt agcgaattca
     1501 tgggaaagtc atatccaact gtcagcgcag actatcagga tgcagttgaa aaggccaaaa
     1561 agaaattacg tggattcatt gcagagaaac gttgcgcccc tttgatgctg cgtcttgcat
     1621 tccactcggc tgggaccttc gacaagggaa caaagacagg ggggcctttt ggcacaatca
     1681 aacatcccgc ggagttggca cattcagcga acaacggcct tgacatcgct gttcgtttac
     1741 tggagcccct gaaggcggaa tttcctattt taagctatgc ggatttctat cagttagcgg
     1801 gagttgtcgc agttgaagta acaggtggcc caaaggtgcc attccacccg gggcgtgagg
     1861 ataaaccaga accaccgcct gagggtcgct tgccagatcc gacgaagggc agcgaccatt
     1921 tacgcgatgt tttcggaaag gcaatgggat taaccgacca agacattgtc gccctttcgg
     1981 gcgggcatac cattggcgca gcccacaaag agcgcagcgg ttttgaaggc ccttggacca
     2041 gcaatcctct tatctttgac aactcgtatt tcacggaatt actgtcgggg gagaaggagg
     2101 gcttgctgca gctgcctagc gataaggcac tgttaagcga tccggttttc cgtccattag
     2161 tggacaagta tgccgcggac gaagatgcct tttttgcaga ctacgccgag gcgcaccaga
     2221 aattgtcaga gctgggtttc gcggatgcca agcttggcgg tggaggttct ggtggcggtg
     2281 gttcaggagg tggcggatcc aacgccaaag agattgttgt gcacgcctta cgccttctgg
     2341 aaaatggcga cgcacgtggt tggtcagatt tatttcaccc tgagggagtt ctggaatacc
     2401 cgtacccgcc accgggttac aagactcgtt ttgaaggtcg cgaaaccatt tgggcacaca
     2461 tgcgcttgtt tcctgaatac atgaccattc gctttaccga tgtgcagttc tatgagaccg
     2521 ctgatcctga cttagccatc ggtgagtttc atggcgacgg agtgcttacg gcaagtggtg
     2581 gcaaattggc ctatgactat attgcggttt ggcgcacccg tgatgggcag atccttcttt
     2641 atcgtctttt tttcaaccca ttacgtgtgc ttgagccttt gggtggtgtt gaggcagcag
     2701 ctaagatcgt ccagggtgca ggctcagaga atttgtattt ccagggtctc gagcaccacc
     2761 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     2821 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     2881 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     2941 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3001 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3061 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3121 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3181 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3241 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3301 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     3361 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3421 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     3481 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     3541 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     3601 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     3661 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     3721 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     3781 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     3841 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     3901 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     3961 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     4021 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     4081 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     4141 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     4201 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     4261 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     4321 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4381 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4441 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4501 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4561 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4621 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4681 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4741 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4801 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4861 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4921 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4981 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5041 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5101 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     5161 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5221 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     5281 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     5341 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     5401 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     5461 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     5521 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     5581 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     5641 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     5701 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     5761 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     5821 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     5881 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     5941 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     6001 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     6061 acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     6121 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     6181 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     6241 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     6301 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     6361 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     6421 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6481 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gttttt
//
